For cultured cells, cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, stained using anti-Cas and anti-RelA as primary antibodies as well as Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 546 anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as secondary antibodies, and then viewed with a confocal microscope (Nikon A1R system). 4′,6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) was used to stain the nucleus.

For osteocytes in mouse midshaft tibiae, mice were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Tibiae were immersed in the same fixative at 4°C overnight and decalcified in 10% EDTA (pH 7.4) at 4°C for 14 days. The samples were embedded in paraffin, sectioned with 5-μm thickness, and incubated with primary antibodies (anti-Cas, anti-RelA, and anti–acetylated RelA) at 4°C overnight. Alexa Fluor 488–conjugated goat anti-mouse IgG, Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 594 anti-mouse IgG were used as secondary antibodies. Nuclei were counterstained using DAPI. Sections were mounted with Fluorescent Mounting Media (Dako, CA, USA). Quantitative 3D analysis of nuclear/total Cas was conducted using Imaris software (Bitplane, Zurich, Switzerland).

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